The signaling by G protein-coupled receptors (GPCRs) is regulated by receptor phosphorylation and arrestin binding to active phosphoreceptor. Arrestin binding terminates G protein-mediated signaling, tags GPCRs for internalization, and often redirects the signaling to G-protein-independent pathways via c-Src and ERK1/2 and JNK3 activation cascades. Both receptor-bound and free forms of arrestin regulate the function and affect the intracellular localization of multiple signaling molecules. The main objective of this proposal is to elucidate the structural basis of arrestin function as an organizer of multi-protein signaling complexes in the cell. We propose to identify arrestin elements involved in the interactions of both non-visual arrestins in their free and receptor-bound state with non-receptor signaling molecules c-Src, ERK2, JNK3, upstream kinases, and ubiquitin ligase Mdm2. To this end, we propose to use site-directed mutagenesis, direct binding assay, site-directed spin labeling of arrestins and EPR spectroscopy, as well as trafficking assays in living cells. We also propose to reconstruct arrestin-containing "signalosomes", i.e., the arrestin-receptor complexes with protein kinases, from purified components in order to elucidate which proteins bind arrestin directly and what are the functional consequences of the interaction of Src, ERK2, and JNK3 with arrestin-receptor complex. Based on the size of the arrestin molecule and the number of its non-receptor interaction partners, we hypothesize that some (if not all) of these kinases will compete with each other for arrestin binding. We propose to test this hypothesis and measure the affinity of Src, ERK2, and JNK3 for free and receptor-bound arrestin. Identification of arrestin binding sites for non-receptor partners will allow us to construct arrestin mutants with selectively enhanced or disabled interaction sites. Using these mutants for targeted experimental manipulation we propose to study the physiological significance of these interactions in the cell. This information will set the stage for designing peptide and small molecule mimics of the interaction surfaces that can be used as experimental and therapeutic tools.